Branched cationic copolymers and methods for antimicrobial use

ABSTRACT

The invention provides a branched copolymer for the treatment of bacterial, fungal, and viral infections. The branched copolymer is characterized as having (i) at least 10 amino acids, (ii) at least 10% of the amino acids are histidine, (iii) at least 10% of the amino acids are non-histidine, (iv) said branched polymer comprising one or more backbones, (v) one or more terminal branches, and (vi) optionally, one or more non-terminal branches.

1. BACKGROUND OF THE INVENTION

1.1 Field of Invention

The present invention relates generally to antimicrobial agents, moreparticularly to antimicrobial peptides, and most specifically tobranched cationic antimicrobial peptides.

1.2 General Background

Increasing bacterial resistance to conventional antibiotics has spurredresearch for novel antimicrobial agents. One such area concernsantimicrobial peptides (Hancock, 1999; Hancock and Lehrer, 1998, Lehrerand Ganz, 1996). Cationic peptides have an important role in defendingthe host against invading microbial organisms in both plants and animals(Otvos, 2000; Otvos et al., 2000). The activity of some cationicpeptides are restricted to either gram positive or gram negativebacteria while others are active against both. Cationic peptides arealso effective against fungal and viral infection Hancock, 1999).

Cationic peptides are small, 12-35 amino acids, diverse both in sequenceand structure, and often possess a net positive charge due to thepresence of arginine and lysine (Hancock, 1999). Since living organismslack the ability to synthesize branched peptides all cationic microbialpeptides are linear and this limits antimicrobial activity. Four majorclasses of antimicrobial peptides by structure are recognized:β-stranded, e.g. defensins and protegrins; α-helical, e.g. magainins andcecropins; extended coil, e.g. indolicidin and bac 5; and loops, e.g.bacteninin and polymyxins (Hancock, 1997).

1.3 Discussion of Prior Art

Patent literature has described microbial activity by several cationicpeptides: WO 8900199; WO 885826, WO 8604356, EP 193351, EP 85250, U.S.Pat. No. 6,465,429, & U.S. Pat. No. 5,912,230. Helicity, hydrophobicity,and charge are considered important to cationic peptide selectivitytoward prokaryotic membranes (Dathe and Wieprecht, 1999; Hoover et al.,2001; Hughes, 1999).

In addition to cationic amino acids cysteine, proline, glycine,histidine, and hydrophobic amino acids appear to have a structuralfunctional role in selected microbial peptides (Ibid., Epand and Vogel,1999; La Rocca et al., 1999; Oppenheim et al., 1998a; Sitaram andNagaraj, 1999). Histidine, for example, is not present in manyantimicrobial peptides but is found in the saliva in one group of lowmolecular weight linear peptides: Histatin 1 and 3 (Oppenheim et al.,1998b; Sabatini and Azen, 1989; Tsai and Bobek, 1998). Proteolyticfragments of Histatin 1 and 3 have been shown to have antifungalactivity (Ibid.).

Defensins and protegrins are the primary antimicrobial peptides inhumans wherein the former is abundant in phagocytes and small intestinalmucosa. Increases in serum defensins in non-neutropenic patients withsepsis have been observed and it has been suggested that defensins playa role in host defense against severe sepsis (Thomas et al., 2002). Therole antimicrobial peptides have in host protection against systemicinfections is not clear but the role played in prevention and control oflocal infections, particularly in higher organisms, is well evidenced.

Prokaryotes have several properties heightening sensitivity to cationicmicrobial peptides in comparison with eukaryotes. The almost universalnegative charge on cell membranes and walls of bacteria is consideredresponsible for the antibacterial activity of cationic peptides. Thischarge is partly due to certain components: lipopolysaccharide (LPS) andanionic lipids in membranes; peptidoglycans and techoic acid in walls.Eukaryotic cell walls, in contrast, lack anionic lipids. A lack ofcholesterol and lesser potential for membrane transfer also increase thesensitivity of prokaryote bacteria in comparison with eukaryotes and thecombination of these properties enable cationic peptides to target andklll the former. At high concentrations, however, cationic peptides maybe toxic to eukaryotic cells.

Gram negative bacteria have a peptidoglycan layer between the inner andouter cell membranes. Cationic peptides first bind to the negativelycharged LPS in the outer membrane and then bind to the anionic lipids ofthe inner membrane in a self promoting mechanism. Both actions require apositive charge and this also enables penetration of the single innermembrane of gram positive bacteria. A sufficient number of cationicamino acids with a positive charge at physiologic pH is clearlynecessary. Two mechanisms have been suggested for this: formation of atrans-membrane pore; and membrane solubilization. The latter appears tobe the primary mechanism for inhibiting prokaryotic bacterial growth(Bechinger et al., 1999; Oren et al., 1999; Oren eta al., 2002; Oren andShai, 1998) while the provision of a cationic cleavage peptide bylactoferrin (Vogel et al., 2002) supports the former. It has beensuggested, moreover,that these mechanisms are not mutually exclusive andthat not all antimicrobial peptides act by the same mechanism (Ibid.) Inaddition to these prokaryotic membrane destruction mechanismsβ-defensins may enhance host defenses by reducing endotoxin levels(Giacometti et al., 2001) or by interacting with chemokine receptors(Yang et al., 2002).

Some bacteria evidently have outer membranes that are impenetrable bycationic peptides (Hancock, 1997; Preschel and Collins, 2001) and whilesynergism between several different type of antibiotics and cationicpeptides has been shown in pre-clinical models (Hancock, 1999) andIB-367 has been evaluated in phase II clinical trials on oral mucositis(Bellm et al., 2000; Mosca et al., 2000) little is known about acute orchronic toxicity of may cationic peptides administered intravenously.

Defensins are similar to neurotoxic venoms in being small cationicpeptides with cysteine bridges (Bontems et al., 1991; Kourie andShorthouse, 2000) but are dissimilar in not affecting ion channelactivity. Many defensins (Harder et al., 2001; Jia et al., 1999; Mallowet al., 1996) and neurotoxins (Blanc et al., 1996; Cruz et al., 1987;Gilles et al., 2000; Martin and Rochat, 1984) have histidines in closeproximity with cationic amino acids and since the role of histidines inneurotoxins has not been investigated this similarity urges caution indevelopment of cationic peptides de novo.

Non-viral gene therapy carriers are also cationic, often containarginine and lysine, and the resulting positive charge is important toboth interaction with DNA and the cell surface. Branched polymers havebeen made primarily from histidine and lysines (Chen et al., 2001, WO147496) that, in contrast to cationic liposomes, are effective carriersof DNA into cells. The role of histidine in these non-viral gene therapycarriers is thought to be the buffering of endosomes but endosomes haveno role in the antimicrobial activity of cationic peptides.

1.4 Statement of Need

The similarity of defensins to neurotoxins in being linear smallcationic peptides with cysteine bridges has been noted as urging cautionin the development of cationic peptides de novo for use as anantibiotic. Toxicity to cardiac and neural cells is recognized as agrave concern particularly for a systemic antibiotic for both naturaland de novo peptides.

While the prior art has, as related above, demonstrated widespreadsuccess of defensins possessing cationic amino acids in fighting localinfections in vivo and of cationic peptides as an antimicrobial invitro, the development of an effective antimicrobial peptide withminimal toxcity is essential.

The demonstrated ability of bacteria in recent years to developresistance to all known antibiotics, including, most recently and mostalarmingly, strains resistant to vancomycin, the antibiotic of lastresort, is a problem of vast dimension. Bacterial infection has, again,become a primary concern of surgical medicine. And while for obviousreasons the phenomenon has not been advertised, hospitals have becomereservoirs for resistant bacteria. A poignant need is thereforerecognized for an alternative to conventional systemic antibiotics thatwill not incur the development of bacterial resistance.

2. SUMMARY OF THE INVENTION

2.1 Objects of thte Invention

A primary object of the present invention is to provide a model fornon-toxic antimicrobial agents that can be used systemically as analterative to conventional systemic antibiotic, antiviral and antifungaltherapies that do not incur the development of resistance by the targetpathogens. Another object of the present invention is to provide a modelfor non-toxic antimicrobial agents that can be used systemically in genetherapy. Other auxiliary and ancillary objectives of the presentinvention may become apparent in a reading of the principles relating tosaid invention below and the detailed discussion of preferred embodimentfollowing.

2.2 Principles Relating to the Present Invention

In achievement of the objects above it is suggested that cationicpeptides efficacious in killing bacteria without incurring thedevelopment of bacterial resistance be utilized in a form that isnon-toxic. Unexpectedly a branched cationic peptide composed ofhystidine and lysine has more potent antimicrobial activity, and mostunexpectedly no demonstrated toxicity, in comparison with a linearcationic peptide. A transport polymer comprised of a core and branchedpeptides possessing metabolizable peptide-bond linkages to a lysine coreis specifically suggested for minimizing concerns with toxicity,improving antibacterial efficacy, and avoiding the development ofbacterial resistance.

A branched peptide comprised of at least ten amino acid residuescontaining at least ten per cent histidine and at least ten per centnon-histidine amino acid residues is suggested. Cationic amino acidsincluding lysine, arginine and ornithine are suggested for thenon-histidine amino acids. Use of the transport polymer in conjunctionwith a pharmaceutical delivery component including a lipid, amicrosphere, or another polymer, is also suggested. It is specificallysuggested, in the case of a lipid, that a lamellar, uni or multi,liposome have the cationic peptide incorporated therein.

It is suggested that the peptide branches to the polymer beindependently selected from the group of linear or branched polypeptidesderived formulaically from at least one of the following sevensequences:

-   (1) K-H-K-H-K-H-K-G-K-H-K-H-K;-   (2) K-H-K-H-K-H-K-G-K-H-K-H-K-H-K;-   (3) K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K;-   (4) K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-K-G-K-H-K-H-K-K;-   (5) K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K;-   (6) K-K-H-H-H-K-H-H-H-K-K-H-H-H-K-H-H-H-K-K;-   (7) K-H-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K;    wherein conventional single letter amino acid symbols are utilized    with K=lysine, H=histidine, and G=glycine; and specifically    including serial repetitions, reversals, and the following formulas:-   (8) [K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K]_(x+1)[K_(x)] with    1<X<30;-   (9) [K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K]_(x+1)[K_(x)] with    1<X<30;-   (10) [K-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K]_(x+1)[K_(x)] with 1<X<30;    wherein the initial amino acid within the brackets is the N-terminal    amino acid of the polymer or a peptide branched thereto and the last    amino acid within the brackets is the C-terminal end that is    conjugated to the amino groups, α or ε, of the lysine core.

Administration of a pharamaceutical agent delivery composition includingan active agent and a polymer in accordance with the formulae givenabove is also suggested. Bacteria, fungi, and viruses are encompassed asis intracellular infection. Both gram positive and negative bacteria areaddressed. Viruses include enveloped containing viruses includingretroviruses including HOV. Microbial organisms of both the animal andplant kingdoms are encompassed.

3. BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic of the structure of histidine-lysine branchedcopolymers used in antimicrobial and in vivo toxicity studies whereinthe first three polymers are linear and polymers 4-8 are branchesemanating from a core of lysine (K).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

5.1 Definitions

The term ‘amino acid’ is inclusive of the twenty common amino acids aswell as non-standard amino acids including D-amino acids and chemicallyor biologically produced derivatives of common amino acids.

A compound is ‘associated with’ a second compound if the two have formeda complex as a result of covalent or non-covalent interaction betweenthe two.

‘Copolymer’ refers to a polymer containing two or more types of unitsregardless of the arrangement of units or molecular structure. A‘histidine copolymer’ includes histidine as a unit type and comprises a‘transport polymer’ in the present description.

The term ‘peptide’ includes linear, branched, and cyclic amino acidchains including at least two amino acid residues and the term‘polypeptide’ connotes at least two joined peptides.

The term ‘lipid’ includes any chemical species having a hydro phobic orphilic part enabling association with or incorporation into micelles orliposomes. Hydrophilicity typically is derived from the presence ofphosphato, carboxylic, sulfato, amino, sulfhydryl, nitro and similargroups while hydrophobicity is typically conferred by cholesterol, itsderivatives, or inclusion of groups including long chain saturated andunsaturated aliphatic hydrocarbons including substitution with at leastone aromatic, cycloaliphatic, or heterocyclic group.

The term ‘non-cationic lipid’ refers to any of a number of lipid speciesexisting in: an uncharged state, a neutral zwitterionic form, or ananionic form; at physiological pH including diacylphosphatidylcholine,diacylphoshatidylethanolamine, ceramide, sphingomyelin, cephalin,cardiolipin, cerbrosides, DOPE, and cholesterol.

The term ‘cationic lipid’ refers to any of a number of lipid speciescarrying a net positive charge including DODAC, DOTMA, DDAB, DOSPER,DOSPA, DOTAP, DC-Chol and DMRIE. A number of commercial preparations areavailable including: LIPOFECTIN® and LIPOFECTAMINE both from GIBCO/BRL,Grand Island, N.Y., USA; TRANSFECTAM® from Promega Corp., Madison, Wis.,USA.

The term ‘delivery component’ connotes any stabilizing agent forcationic peptides in vivo or aids in the delivery of cationic peptidesto an infectious organism including incorporation of cationic peptidesinto liposomes and ligands including PEG prolonging half life in blood.

An ‘additive therapeutic infectious agent’ is an adjunct to a cationicpeptide delaying, preventing, or reducing the severity of infectiousdisease, i.e. reducing infection, including antibiotics and nucleicacids including RNA inhibitors effective against reiroviruses.

5.2 Pharmaceutical Agency

A branched cationic polypeptide polymer in accordance with theprinciples relating to the present invention comprised of histidine andother amino acid residues can be used alone or in association with adelivery component as a pharmaceutical agent preferably by incorporatingthe former into a stable complex with the latter and further preferablyprovided in a suitable pharmaceutically acceptable carrier. Saidbranched cationic polypeptide polymers can also be used alone as, or inassociation with, an additive therapeutic infections agent with orwithout an intracellular delivery component inclusive of pegylation,selected substitution of L-amino to D-amino acids, cell specificligands, microspheres, lipids, and various lipid based substancesincluding liposomes and micelles. A stabilizing compound covalentlyattached to either lipids or to cationic peptides can also be added.Polyethlene glycol is an example.

Preferred lipids form liposomes in a physiologically compatibleenvironment including, for example, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, dilinoleoylphosphatidylcholine. Lipidswith a molarity of five to fifty per cent can be used with helper lipidssuch as cholesterol to increase stability in the bloodstream. Andpegylated lipids in molar perentages of 0.05 to 0.5 can be used toprolong in vivo half life of the liposomes.

An additive therapeutic agent suitably comprises any agent that togetherwith a branched cationic polypeptide polymer in accordance with theprinciples relating to the present invention demonstrates reduction inthe infectious organism including, as example, antibiotics and nucleicacids administered separately from said branched cationic polypeptidepolymer or in complex therewith by covalent or non-covalent bonding.Aminoglycosides, penicillin, cephalosporins, fluroquinolones,carbepenems, tetracylcines, and macrolides are examples of antibioticsthat are considered useful for synergistic therapy with a branchedcationic polypeptide polymer in accordance with the principles relatingto the present invention. Plasmid based therapies, antisense, ribozymes,DNAzymes and RNA inhibitors provide examples of useful additivetherapeutic infectious agents while RNAi in complex with said polymer ispreferred.

5.3 Structure

A branched cationic polypeptide polymer in accordance with theprinciples relating to the present invention comprised of histidine andother amino acid residues preferably contains about twenty to threehundred said residues, more preferably thirty to a hundred, and mostpreferably thirty to seventy amino acid residues. The percentageconstituency of histidine is preferably ten to ninety, more preferablytwenty to eighty, and most preferably forty to seventy and arepreferably interspersed uniformly into the transport polymer structurewith at least one histidine residue in every segment of two to fiveamino acid residues. More preferably the distribution is at least onehistidine residue in every segment of two to three and most preferablyone in every two amino acid residues. Distribution of one to fivehistidine residues in every segment of two to five amino acid residuesis suggested while one to four histidine residues in every segment oftwo to four amino acid residues is preferred. Non histidine residues arepreferably distributed to achieve an average of one non-histidineresidue in every segment of two to seven amino acid residues.

Preferred embodiment has the polymer branches bonded to a lysine coreeach comprised of one of the following formulas:

-   (1) K-H-K-H-K-H-K-G-K-H-K-H-K;-   (2) K-H-K-H-K-H-K-G-K-H-K-H-K-H-K;-   (3) K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K;-   (4) K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-K-G-K-H-K-H-K-K;-   (5) K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K;-   (6) K-K-H-H-H-K-H-H-H-K-K-H-H-H-K-H-H-H-K-K;-   (7) K-H-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K;    wherein conventional single letter amino acid symbols are utilized    with K=lysine, H=histidine, and G=glycine; and specifically    including serial repetitions and reversals.

A more preferred embodiment has the polymer branches bonded to a lysinecore each comprised of one of and the following formulas:

-   (8) [K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K]_(x+1)[K_(x)] with    1<X<30;-   (9) [K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K]_(x+1)[K_(x)] with    1<X<30;-   (10) [K-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K]_(x+1)[K_(x)] with 1<X<30;    wherein the initial amino acid within the brackets is the N-terminal    amino acid of the polymer or a peptide branched thereto and the last    amino acid within the brackets is the C-terminal end that is    conjugated to the amino groups, α or ε, of the lysine core. In most    preferred embodiment X ranges from three to five.

The backbone peptide or core of a branched cationic polypeptide polymerin accordance with the principles relating to the present invention canbe constituted of any branching amino acid residue, preferably lysine,and between one and thirty peptide branches covalently attached tobranching amino acid residues of the backbone peptide directly orthrough non-branching amino acid residues. The covalent bonding includespolypeptide, ester, and disulfide bonds.

Branching amino acids preferably possess a free amino side chain group:e.g. diaminobutyric acid, lysine, arginine, ornithine, diaminopropionicacid and citrulline; but can alternatively possess a free carboxyl sidechain group: e.g. glutamic acid, aspartic acid, and homocitrulline.

A branched cationic polypeptide polymer in accordance with theprinciples relating to the present invention may also comprise apolypeptide-‘synthetic monomer’ copolymer wherein the transport polymerbackbone comprises covalently linked segments of polypeptide andsynthetic, preferably bio-compatible, monomer or polymer. Suitablemonomers as well as methods for preparing a polypeptide-‘syntheticmonomer’ copolymer are described in U.S. Pat. No. 4,511,478 for‘Polymerizable Compounds and Method for Preparing Synthetic Polymersthat Integrally Contain Polypeptides’ by Nowinski et al. incorporated byreference herein.

5.3.1 Methods of Manufacture

Polypeptides for use in manufacturing branched cationic polypeptidepolymers in accordance with the principles relating to the presentinvention can be chemically synthesized and purified by techniques wellknown in the art. For example, by employing theN-α-9-fluorenylmethyloxycarbonyl or Fmoc solid phase polypeptidesynthesis chemistry using a Rainin Symphony Multiplex PolypeptideSynthesizer.

Branched cationic polypeptide polymers in accordance with the principlesrelating to the present invention are made by including one or moreamino acids within the amino acid sequence with a free side chaincapable of forming a polypeptide bond with one or more amino acids, andthus capable of forming a ‘branch’, and reacting with a side chain tothat locus. This can be done by any method for covalently lihking anynaturally occurring or synthetic amino acid to any naturally occurringor synthetic amino acid in a polypeptide chain which has a side chaingroup able to react with the amino or carboxyl group on the amino acidsso as to become covalently attached to the polypeptide chain.

In particular, amino acids with a free amino side chain group including,as example, diaminobutyric acid, lysine, arginine, ornithine,diaminopropionic acid, and citrulline, can be incorporated into apolypeptide so that an amino acid can form a branch therewith by, forexample, forming a polypeptide bond to the free amino side group fromthat residue. Alternatively, amino acids with a free carboxyl side chaingroup including, as example, glutamic acid, asparitic acid, andhomocitrulline, can be incorporated into the polypeptide so that anamino acid can form a branch therewith, for example, by forming apolypeptide bond to the free carboxyl side group, from that residue. Theamino acid forming the branch can be linked to a side chain group of anamino acid in the polypeptide chain by any type of covalent bondincluding polypeptide bonds, ester bonds and disulfide bonds.

The best known manner of preparation of branched cationic polypeptidepolymers in accordance with the principles relating to the presentinvention follows.

For example, but not by way of limitation, branched polypeptides can beprepared as follows: (1) synthesize peptides on a Symphony multiplepeptide synthesizer at a 0.025 mM scale using Fmoc(9-fluorenylmethyloxycarbonyl) chemistry, (2) remove Fmoc group(deprotection) with 20% piperidine/0.1M HOBt by incubating 3 times for20 min; (3) following deprotection, wash resin 6 times for one min withDMF/DCM (1:1; primary solvent); (4) mix Fmoc-lysine (Dde) with HATU/DIEAin a 1:1:1.5 molar ratio; (5) mix activated amino acid with resin for 45min at RT; (6) wash 3 times for 1 min with DMF/DCM; (7) cap anyuncoupled free N-terminal amino groups on the resin using 50% aceticanhydride in DMF. Wash with capping reagent two times for 15 min; (8)wash 3 times for 1 min with the primary solvent; (9) repeat cycles ofsteps 2-8 to couple additional Fmoc-Lys(Dde) residues, dependent on thenumber of branches; (10) following the capping step after addition ofthe final Fmoc-Lys(Dde), remove Dde groups by incubation of the peptideresin in 2% hydrazine for 30 min; (11) wash peptide resin 6 times for 1min with the primary solvent; (12) to add amino acids and to synthesizebranches to the lysine core, remove Fmoc group (deprotection) byincubating 3 times for 20 min with 20% piperidine containing 0.1M HOBt;(13) following deprotection, wash the resin 6 times for one min withDMF/DCM (1:1; primary solvent); (14) catalyze amino acid coupling withHATU/DIEA as the activator. Mix Fmoc-Lysine(Boc), with HATU/DIEA in a1:1:1.5 molar ratio; (15) mix activated amino acid with resin for 45 minat RT; wash 3 times for 1 min with the primary solvent; (16) cap anyuncoupled free N-terminal amino groups on the resin using 50% aceticanhydride in DMF. Wash resin with 50% acetic anhydride in DMF two timesfor 15 min; (17) wash 3 times for 1 min with the primary solvent; (18)repeat steps a) through g) for each successive amino acid additionperformed until the desired polymer is completed (a) remove Fmoc group(deprotection) by incubating 3 times for 20 min with 20% piperidinecontaining 0.1M HOBt. b) following deprotection, wash the resin 6 timesfor one mnin with DMF/DCM, c) Catalyze amino acid coupling withHATU/DIEA as the activator. Mix Fmoc-Lysine(Boc), with HATU/DIEA in a1:1:1.5 molar ratio, d) Mix activated amino acid with resin for 45 minat RT, e) wash 3 times for 1 min with the primary solvent, f) cap anyuncoupled free N-terminal amino groups on the resin using 50% aceticanhydride in DMF. Wash resin with 50% acetic anhydride in DMF two timesfor 15 min, and g) wash 3 times for 1 min with the primary solvent.)(19) cleave, purify, and analyze branched polymer, Branched polypeptidesprepared by this method will have a substitution of lysine at the aminoacid position, which is branched. Branched polypeptides containing aminoacid analog substitution (e.g., diaminobutyric acid) can be preparedanalogously to the procedure described above, using the t-Boc coupledform of the amino acid or amino acid analog.

5.4 Methods of Using the Pharmaceutical Agent

The invention comprises a method for delivering a pharmaceutical agentto an infectious agent. These agents may treat gram positive, gramnegative, fungi, or viruses. Preferably, the compositions areadministered to animals, including humans by injection. Injection may besystemic (by i.v. for systemic infections) or local (for example,injection to the site of a localized injection such a cellulitis or anabcess; aerosolized therapy for lung infections). ln another preferredmethod of therapy, the pharmaceutical agent may be used as prophylacticsprior to eye, cardiac, orthopedic, vascular, or pelvic surgery. Theamount of polymer administered depends on a variety of factors known inthe art, for example, the desired effect, subject state, etc., and canreadily be determined by one skilled in the art.

Examples of gram positive bacteria treatable with the compositions andmethods includes: Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophytics, Streptococcus pneumonia, Streptococcusenterococcus, Streptococcus pyogenes, Listeria monocytogenes Clostridiumbotulinum, Clostridium tetani, Clostridium difficile, Clostridiumperfringens, Bacillus anthracis, Bacillus cereus, Bacillusthuringiensis, Bacillus mycoides, Bacillus subtilis, Micrococcus luteus,Mycobacterium tuberculosis, Peptococcus niger, Enterococcus faecalis,Enterococcus faecium, Erysipelothrix rhusiopathiae, Nocardia asteroides,Nocardia brasiliensis, Actinomyces actinomycetemcomitans, Actinomycesviscosus, and Actinomyces israeli.

Examples of gram negative bacteria treatable with the compositions andmethods includes: Escherichia coli, Klebsiella pnuemoniae, Pseudomonasaeruginosa, Pseudomonas mallei, Pseudomonas melioidosis, Pseudomonaspseudomallei, Bacteroides fragilis, Bacteroides Thetaiolaomicron,Bacteroides melaninogeizicus, Bacteroides distasonis, Prevotella bivia,Prevotella disiens, Haemophilus influenzae, Haemophilus parainfluenzae,Haemophilus ducrey, Haemophilus aegypticus, Niesseria meningitidis,Niesseria gonorrhoeae, Legionella pneumophila, Salmonella enteritidi,Shigella dysenteriae, Proteus mirabilis, Proteus mirabilis, Enterobacteraerogenes, Enterobacter, cloacae, Enterobacter hafniae, Serratiamarcecens, Citrobacter freundii, Yersinia pestis, Yersiniaenterocolitica, Vibrio cholerae, Brucella abortus, Brucella melitensis,Brucella suis, Brucella canus, Bordetella pertussis, Bordetellaparapertussis, Bordetella bronchiseptica, Campylobacter jejuni,Hellobacter pylori and Rickettsia rickettsii

Examples of fungi treatable with the compositions and methods includes:Candida Albicans, Cryptococcus neoformans, Aspergillus fumigatus,Aspergillus flavus, Aspergillus niger, Trichophyton rubrum, Trichophytonmentagrophytes, Microsporon lanosum, Microsporon canis, Microsporonaudouini, Epidermophyton floccosum, Blastomyces dermatitidis,Coccidioides Immitis, and Histoplasma capsulatum.

Examples of viruses treatable with the compositions and methods includeviruses containing membrane envelopes including retroviruses (e.g.,HIV).

EXAMPLES

The polymers in the Examples are based on histidine and lysine (orarginine) copolymers. Cationic amino acids are known to interact withthe negatively charged membranes of bacteria or viruses (e.g.,retroviruses). The data presented herein is consistent with the ideathat both the cationic amino acid and histidine component of thecopolymer and branching of the copolymer aid in the destruction of thebacteria.

6.1 Materials

Bacteria; A gram negative bacteria E. coli transformed with akanamycin-resistant plasmid was used in these experiments and grown inLB media (Biofluids, Rockville, Md.).

Polymers: The biopolymer core facility at the University of Marylandsynthesized the polymers on a Ranim Voyager synthesizer (PTl, Tuscon,Az.). The polymers were then purified on an HPLC (Beckman, Fullerton,Calif.) and analyzed with mass spectroscopy (Perseptive Biosystems,Foster City, Calif.) to verify the predicted molecular mass. Measurementof histidine copolymers with poly-L-lysine (Sigma Co., St. Louis, Mo.)used as a standard were done with2,6-dinitro-4-trifluoromethylbenzenesulfonate (Pierce Co., Rockford,Ill.) as previously described. The following polymers were made: 1) R-H(19mer) [R-B-R-H-R-H-R-H-R-G-R-H-R-H-R-H-R-H-R] (SEQ ID NO: 8); 2) H-K(19mer) [K-H-K-H-K-H-K-K-G-K-H-K-H-K-H-K-H-K] (SEQ ID NO:3); 3) HK-R(19-mer) [R-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K] (SEQ ID NO: 9); 4)H-K4b (79-mer) [K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K]₄K₃ (SEQ ID NO:10); 5) H²K2b (41-mer) [K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K]₂K (SEQID NO: 11); 6) H²K4b (83-mer) [K-H-K-H-H-K-H-H-K-H-H-K-H-K-H-H-K-H-K]₄K₃(SEQ ID NO: 12); H³K4b (71-mer) [K-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K]₄K₃(SEQ ID NO: 13); 7) H³ KN4b (71-mer)[K-H-H-H-N-H-H-H-K-H-H-H-K-H-H-H-K]₄K₃ (SEQ ID NO: 14). Note that 10through 13 are branched polymers in which polymer is attached to the αand/or ε amino groups of the lysine residue and the central core oflysines are linked to one another by peptide bonds (FIG. 1).

6.2 In Vitro Bacteria Inhibition Studies:

For in vitro studies, several polymers were tested for their ability toinhibit gram negative bacteria The linear polymers (HK, SEQ ID NO: 3;and RH, SEQ ID NO: 8) and the minimally branched HK (H²K2b, SEQ ID NO:11) showed only moderate reduction on bacteria growth. The highlybranched polymers (H²K4b, SEQ ID NO: 12; and H³K4b, SEQ ID NO: 13)showed the most antimicrobial activity against a kanamycin-resistantbacteria. The histidine and lysine ratio and their sequence order havean important role in the-polymers' effects. The following conclusionscan be made:

-   1) the polymer with greatest number of lysines (e.g., HK4b, SEQ ID    NO: 10) did not show the highest antimicrobial activity. This    indicates that here is another factor besides the interaction of the    positively charged amino acids with the negatively charged membranes    of the bacteria.-   2) the polymer, H³KN4b (SEQ ID NO: 14) with the most histidines and    the highest histidine:lysine ratio showed moderate antimicrobial    activity when compared to H²K4b (SEQ ID NO: 12) and H³K4b (SEQ ID    NO: 13). Since it is likely that H³KN4b has the greatest ability to    chelate key transitional elements, chelation of key elements in LB    media, separate from the polymer's role of binding to the bacteria    membrane, is unlikely to have a major role in the inhibition of    bacteria.-   3) The sequence order of lysine and histidine in the branched    polymer has a major role in the inhibition of bacteria. Altering    these sequences affects the antibacterial activity of the bacteria.    Sufficient cationic amino acids are critical for binding whereas the    exact function of histidine is unclear. Data supports that cationic    amino acids need to be evenly distributed in branches, particularly    at N-terminal ends. Too many cationic amino acids in the polymer,    however, reduce antibacterial activity.-   4) Histidine may have a role in the interruption of iron transport    systems and/or it may enhance the hydrophobic interactions of the    polymer with the bacteria membrane. Regardless of the antibacterial    mechanism of histidine within the polymer, lowering the histidine:    lysine ratio significantly reduces antibacterial activity.

5) Linear Polymers inhibit bacteria significantly, less than branchedpolymers. TABLE 1 MIC of HK and RK Polymere Polymers MIC 1. linearHK >300 μg/ml 2. linear RH >300 μg/ml 3. H²K2b (2 branches) >300 μg/ml4. HK4b (4 branches) 198 μg/ml 5. H²K4b (4 branches) 58 μg/ml 6. H²K4b(4 branches) 110 μg/ml 7. H³KN4b (4 branches) 250 μg/mlTurbidity of bacteria (O.D.-600 nm) measured after incubation for 12 hat 37° C. with various polymers and compared to bacterial-containingmedium without polymer. Several concentrations of polymers (300, 150,100, 50 and 25 μg) were added to the 1-ml of LB broth containingbacteria. Polymer numbers 4 to 7 have an increasing higher content ofhistidine compared to lysine, but the# same degree of branching. The MIC is the concentration of theinhibitor in the medium in which bacteria growth is completelyinhibited. Experiment has been repeated 3 times. HK4b, the polymer withthe most lysines, does not have the most activity in reducing bacteriagrowth. Also, H³KN4b, the polymer with most histidines and the highesthistidine:lysine ratio, is not the most active.

6.3 In vitro Toxicity

In vitro toxicity of these polymers was then examined on a malignantcell line (MDA-MB-435). In this cell line, the most effectiveantimicrobial HK polymers showed the least toxicity (as measured by cellnumber) (see Table 2). Unexpectantly, the linear HK polymer wassignificantly more toxic to MDA-MB-435 cells compared to its branchedcounterparts. In contrast, H³K4b had no observed toxicity in these invitro studies. TABLE 2 Reduction In Cell Number After Incubation withPolymers Polymer 150 μg/ml 100 μl/ml 50 μg/ml linear HK 65% 68% 41% H²K4b 22% 12% 5% H³K4b  0%  0% 0%Incubation of 3 polymers at 3 concentrations (150, 100, 50 μg/ml) for 6h in DMEM/10% serum containing MDA-MB-435 cells. 24 h later, cellnumbers were counted for each polymer condition and compared tountreated# cells. This experiment was done twice and each condition has been donein duplicate per experiment. Similar results were observed when polymerswere incubated with MDA-MB-435 cells in the presence of DMEM withoutserum.

6.4 In Vivo Studies:

One of the major limitations of cationic peptides is their potenitialtoxicity when administered systemically. As a result, we tested theacute toxicity of these polymers after injecting them IV. Interestingly,the polymers (branched polymers) that were the most active againstbacteria in vitro showed no evidence of toxicity in vivo (Table 3).Furthermore, mice who received three-600 μg dosages of the H³K4b polymer(each separated by 4 h) showed no evidence of toxicity (e.g., nodecreased activity or paleness). Conversely, linear polymers that hadminimal to moderate antibacterial properties had the greatest in vivotoxicity. The most toxic of the linear polymers was the linear polymer,HR. Even if one arginine adjacent to the histidine residue replaces theamino terminal lysine in the HK polymer (e.g., HK-R), the polymer wasstill very toxic when administered intravenously. The in vivo toxicityof linear HK polymers from most to least is as follows: HR>HK-R>>HK. Formice who died from polymers (HR, HK-R, and HK) at higher dosages,histology of major organs showed no evidence of pathology. There wereparallels between in vitro and in vivo toxicity studies (Tables 2 and3). That is, linear polymers demonstrated the most toxic in thesestudies. The data is consistent with linear polymers interacting andinhibiting ionic channels of the cardiovascular system (similar toneurotoxins). Intravenous injection of linear polymers with arginine orlysines adjacent (or near) to histidines should be avoided. In contrast,mice who received large dosages of the branched HK polymers injectedi.v. showed no untoward effects. TABLE 3 Acute In Vivo Toxicity Polymers600 μg 300 μg 150 μg 75 μg HR 0/2 0/2 2/2 (+++, +) 2/2 (+) HK-R 0/2 0/22/2 (+, +) 2/2 HK 0/2 2/2 (+++, +++) 2/2 (++, +) 2/2 HK4b 2/2 2/2 2/22/2 H²K4b 2/2 2/2 2/2 2/2 H³K4b 2/2 2/2 2/2 2/2Numerator denotes the number of mice who survive treatment, anddenominator denotes number of mice in each treatment group. Each mousewho survived injection but had side effects was defined by the followingscale:+++, inactive for at least 1 min, extreme pallor,++, moderate reduction in activity for at least 1 min;+, moderate reduction in activity in for less than 1 min.

Many of these polymers were initially developed as gene therapy carriersof nucleic acids. Although the nucleic acids mitigated the toxicreactions of the above polymers when administered i.v., the polymers incomplex with nucleic acids showed parallels in toxicity when thepolymers alone were administered i.v.

6. REFERENCES

Throughout this specification various patent and non-patent referenceshave been mentioned. The entire disclosure of each such reference isincorporated herein by reference, as is the entire disclosure of each ofthe following references, to the extent relevant to making and using theintervention as claimed:

-   Bechinger, B., Kinder R., Helmle, M., Vogt, T. C., Harzer, U. and    Schinzel, S. (1999). Peptide structural analysis by solid-state NMR    spectroscopy. Biopolymers 51, 174-90.-   Bellm, L., Lehrer, R. I. and Ganz, T. (2000). Protegrins: new    antibiotics of mammalian origin. Expert Opin. Investig Drugs 9,    1731-42.-   Blanc, E., Fremont, V., Sizun, P., Meunier, S., Van Rietschoten, I.,    Thevand, A., Bernassau, J. M. and Darbon, H. (1996). Solution    structure of P01, a natural scorpion peptide structurally analogous    to scorpion toxins specific for apamin-sensitive potassium channel.    Proteins 24, 359-69.-   Bontems, F., Roumestand, C., Gilquin, B., Menez, A. and Toma, F.    (1991). Refined structure of charybdotoxin; common motifs in    scorpion toxins and insect defensins. Science 254, 1521-3.-   Chen, Q. R., Zhang, L., Stass, S. A. and Mixson, A. J. (2001).    Branched co-polymers of histidine and lysine are efficient carriers    of plasmids. Nucleic Acids Res. 29, 1334-1340.-   Cruz, L. J., Johnson, D. S. and Olivera, B. M. (1987).    Characterization of the omega-conotoxin target. Evidence for    tissue-specific heterogencity in calcium channel types. Biochemistry    26, 820-4.-   Dathe, M. and Wieprecht, T. (1999). Structural features of helical    antimicrobial peptides: their potential to modulate activity on    model membranes and biological cells. Biochim Biophys Acta 1462,    71-87.-   Epand, R. M. and Vogel, H. J. (1999). Diversity of antimicrobial    peptides and their mechanisms of action. Biochim Biophys Acta 1462,    11-28.-   Giacometti, A., Cirioni, O., Ghiselli, R., Viticchi, C.,    Mocchegianzi, F., Riva, A., Saba, V. and Scalise, G. (2001). Effect    of mono-dose intraperitoneal ecropins in experimental septic shock.    Crit Care Med 29, 1666-9.-   Gilles, N., Krimm, I., Boust, F., Froy, O., Gurevitz, M.,    Lancelin, J. M. and Gordon, D. (2000). Structural implications on    the interaction of scorpion alpha-like toxins with the sodium    channel receptor site inferred from toxin iodination and    pH-dependent binding. J Neurochem 75, 1735-45. Hancock, R. E.    (1997). Peptide antibiotics. Lancet 349, 418-22.-   Hancock, R. E. (1999). Host defense (cationic) peptides: what is    their future clinical potential? Drugs 57, 469-73.-   Hancock, R. E. and Lehrer, R. (1998). Cationic peptides: a new    source of antibiotics. Trends Biotechnol 16, 82-8.-   Harder, J., Bartels, J., Christophers, E. and Schroder, I. M.    (2001). Isolation and characterization of human beta-defensin-3, a    novel human inducible peptide antibiotic. J Biol Chem 276, 5707-13.-   Hoover, D. M., Chertov, O. and Lubkowski, J. (2001). The structure    of human beta-defensin-1: new insights into structural properties of    beta-defensins. J Biol Chem 276, 39021-6.-   Hughes, A. L. (1999). Evolutionary diversification of the mammalian    defensins. Cell Mol Life Sci 56, 94-103.-   Jia, H. P., Mills, J. N., Barahmand-Pour, F., Nishimura, D.,    Mallampali, R. K., Wang, G., Wiles, K., Tack, B. F., Bevins, C. L.    and McCray, P. B., Jr. (1999). Molecular cloning and    characterization of rat genes encoding homologues of human    beta-defensins. Infect Immun 67, 4827-33.-   Kourie, J. I. and Shorthouse, A. A. (2000). Properties of cytotoxic    peptide-formed ion channels. Am J Physiol Cell Physiol 278,    C1063-87.-   La Rocca, P., Biggin, P. C., Tieleman, D. P. and Sansom, M. S.    (1999). Simulation studies of the interaction of antimicrobial    peptides and lipid bilayers. Biochim Biophys Acta 1462, 185-200.-   Lehrer, R. I. and Ganz, T. (1996). Endogenous vertebrate    antibiotics. Defensins, protegins, and other cysteine-rich    antimicrobial peptides. Ann N Y Acad Sci 797, 228-39.-   Mallow, E. B., Harris, A., Salzman, N., Russell, J. P.,    DeBerardinis, R. J., Ruchelli, E. and Bevins, C. L. (1996). Human    enteric defensins. Gene structure and developmental expression. J    Biol Chem 271, 4038-45.-   Martin, M. F. and Rochat, H. (1984). Purification of thirteen toxins    active in mice from the venom of the North African scorpion Buthus    occitamus tunetanus. Toxicon 22, 279-91.-   Mosca, D. A., Hurst, M. A., So, W., Vinjar, B. S., Fujii, C. A. and    Falls, T. J. (2000). IB-367, a protegrin peptide with in vitro and    in vivo activities against the microflora associated with oral    mucositis. Antimicrob Agents Chemother 44, 1803-8.-   Oppenheim, F. G., Xu, T., McMillian, F. M., Levitz, S. M.,    Diamond, R. D., Offner, G. D. and Troxler, R. F. (1988b). Histatins,    a novel family of histidine-rich proteins in human parotid    secretion. Isolation, characterization, primary structure, and    fungistatic effects on Candida albicans. J Biol Chem 263, 7472-7.-   Oppenheim, F. G., Xu, T., McMillian, F. M., Levitz, S. M.    Diamond, R. D., Offner, G. D. and Troxler, R. F. (1988a). Histatins,    a novel family of histidine-rich proteins in human perotid    secretion. Isolation, characterization, primary structure, and    fungistatic effects on Candida albicans. J Biol Chem 263, 7472-7.-   Oren, Z., Lerman, J. C., Gudmundsson, G. H., Agerberth, B. and    Shai, Y. (1999). Structure and organization of the human    antimicrobial peptide LL-37 in phospholipid membranes: relevance to    the molecular basis for its non-cell-selective activity. Biochem J    341, 501-13.-   Oren, Z., Ramesh, J. Avrahami, D., Suryaprknsh, N., Shai, Y. and    Jelinek, R. (2002). Structures and mode of membrane interaction of a    short alpha helical lyric peptide and its diastereomer determined by    NMR, FTIR, and fluorescence spectroscopy. Eur J Biochem 269,    3869-80.-   Oren, Z. and Shai, Y. (1998). Mode of action of linear amphipathic    alpha-helical antimicrobial peptides. Biopolymers 47, 451-63.-   Otvos, L., Jr. (2000). Antibacterial peptides isolated from insects.    J Pept Sci 6, 497-511. Otvos, L., Jr., Bokonyi, K., Varga, L.,    Otvos, B. L., Hoffmann, R., Ertl, H. C., Wade, J. D., McManus, A.    M., Craik, D. J. and Bulet, P. (2000). Insect peptides with improved    protease-resistance protect mice against bacterial infection.    Protein Sci 9, 742-9.-   Peschel, A. and Collins, L. V. (2001). Staphylococcal resistance to    antimicrobial peptides of mammalian and bacterial origin. Peptides    22, 1651-9.-   Sabatini, L. M. and Azen, E. A. (1989). Histatins, a family of    salivary histidine-rich proteins, are encoded by at least two loci    (HIS1 and HIS2). Biochem Biophys Res Commun 160, 495-502.-   Sitaram, N. and Nagaraj, R. (1999). Interaction of antimicrobial    peptides with biological and model membranes; structural and charge    requirements for activity. Biochim Biophys Acta 1462, 29-54.-   Thomas, N. J., Carcillo, J. A., Doughty, L. A., Sasser, H. and    Heine, R. P. (2002). Plasma concentrations of defensins and    lactoferrin in children with severe sepsis. Pediatr Infect Dis J21,    34-8.-   Tsai, H. and Bobek, L. A. (1998). Human salivary histantins:    promising anti-fungal therapeutic agents. Crit Rev Oral Biol Med 9,    480-97.-   Vogel, H. J., Schibli, D. J., Jing, W., Lohmeier-Vogel, E. M.,    Epand, R. F. and Epand, R. M. (2002). Towards a structure-function    analysis of bovine lactoferricin and related tryptophan- and    arginine-containing peptides. Biochem Cell Biol 80, 49-63.-   Yang, D., Biragyn, A., Kwak, L. W. and Oppenheim, J. J. (2002).    Mammalian defensins in immunity: more than just microbicidal. Trends    Immunol 23, 291-6.

1. A polymer for treating bacteria, viral, or flngal infectionscomprising a branched polymer with a backbone of 1 or more amino acidresidues and at least one branch with at least 10 amino acid residues,wherein at least 10% of the amino acid residues are histidine and atleast 10% of the amino acid residues are non-histidine.
 2. The polymerof claim 1, wherein at least 30% of amino acids of said peptide arehistidines.
 3. The polymer of claim 1, wherein at least 50% of aminoacids of said peptide are histidines.
 4. The polymer of claim 1, whereinat least 70% of amino acids of said peptide are histidines.
 5. Thepolymer of claim 1, wherein at least 20% of non-histidine amino acidresidues are selected from a group consisting of amino acids which carrya positive charge at physiological pH.
 6. The polymer of claim 5,wherein said group are lysines.
 7. The polymer of claim 1, comprising asubsegment of amino acid residues selected from the group consisting of:(SEQ ID NO:1) K-H-K-H-K-H-K-G-K-H-K-H-K, (SEQ ID NO:2)K-H-K-H-K-H-K-G-K-H-K-H-K-H-K, (SEQ ID NO:3)K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K, (SEQ ID NO:4)K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K-G-K-H-K- H-K-H-K-H-K, (SEQ IDNO:5) K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K, (SEQ ID NO:6)K-K-H-H-H-K-H-H-H-K-K-H-H-H-K-H-H-H-K-K, (SEQ ID NO:7)K-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K,

end-to-end repeats of one or more of the above sequences, and thereverse of any of the above sequences.
 8. The polymer of claim 7selected from the group consisting of:[K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K]₄K₃,[K-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K]₄K₃.
 9. The polymer of claim 1,further comprising at least one delivery component.
 10. The polymer ofclaim 9, whereby delivery component of claim 9 comprises a lipid. 11.The polymer of claim 1, wherein the disorder is septic shock.
 12. Thepolymer of claim 1, further comprising administering an additivetherapeutic infectious agent, whereby said therapeutic agent is selectedfrom the group consisting of antibiotics and/or nucleic acids; andoptionally a delivery component.
 13. The therapeutic composition ofclaim 12, comprising a RNAi nucleic acid that targets viral infections.14. A method for treating bacteria, flngal, or viral infectionsinjections with the said polymer of claim
 1. 15. The method of claim 14,wherein at least 30% of amino acids of said peptide are histidines. 16.The method of claim 14, wherein at least 50% of amino acids of saidpeptide are histidies.
 17. The method of claim 14, wherein at least 70%of amino acids of said peptide are histidines.
 18. The method of claim14, wherein at least 20% of non-histidine amino acid residues areselected from a group consisting of amino acids which carry a positivecharge at physiological pH.
 19. The method of claim 18, wherein saidgroup are lysines.
 20. The method of claim 14, wherein said peptidecomprises a subsegment of amino acid residues selected from the groupconsisting of: (SEQ ID NO:1) K-H-K-H-K-H-K-G-K-H-K-H-K, (SEQ ID NO:2)K-H-K-H-K-H-K-G-K-H-K-H-K-H-K, (SEQ ID NO:3)K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K, (SEQ ID NO:4)K-H-K-H-K-H-K-H-K-G-K-H-K-H-K-H-K-H-K-G-K-H-K- H-K-H-K-H-K, (SEQ IDNO:5) K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K, (SEQ ID NO:6)K-K-H-H-H-K-H-H-H-K-K-H-H-H-K-H-H-H-K-K, (SEQ ID NO:7)K-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K,

end-to-end repeats of one or more of the above sequences, and thereverse of any of the above sequences.
 21. The method of claim 20,wherein said peptide is selected from the group consisting of:[K-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-H-K-H-K]₄K₃,[K-H-H-H-K-H-H-H-K-H-H-H-K-H-H-H-K]₄K₃.
 22. The method of claim 14,further comprising at least one delivery component.
 23. The method ofclaim 22, wherein the delivery component comprises a lipid.
 24. Themethod of claim 14, wherein the disorder is septic shock.
 25. The methodof claim 14, further comprising administering an additive therapeuticinfectious agent, whereby said therapeutic agent is selected from thegroup consisting of antibiotics and/or nucleic acids; and optionally adelivery component.
 26. The method of claim 25, comprising a RNAinucleic acid that targets viral infections.
 27. The method of claim 14for treating bacteria, fungal, or viral infections, wherein theinfection is local or systemic.